# Biogenesis of cyclic and phospholipid-linked enterobacterial common antigen

> **NIH NIH R01** · TEXAS A&M UNIVERSITY · 2024 · $72,022

## Abstract

PROJECT SUMMARY
Nearly 3 million antibiotic resistant infections occur per year in the United States. This problem is especially acute
in gram-negative bacteria, where the outer membrane (OM) which surrounds the aqueous periplasm acts as a
permeability barrier capable of excluding many antibiotics. We are interested in the OM of Enterobacterales (e.g.,
Escherichia, Salmonella, Klebsiella), which are adapted to an enteric environment rich in toxic molecules, such
as bile salts, necessitating an especially strong OM. It has become clear that the permeability of the OM can be
altered by the physiological state of the cell. Specifically, stresses such as nutrient limitation can result in
strengthening of the OM permeability barrier. Elucidation of the pathways responsible for this strengthening will
lead to new targets for the development of small molecules that can weaken the OM permeability barrier. We
have found enterobacterial common antigen (ECA), a conserved component of the Enterobacterales OM and
periplasm, to be important for OM impermeability under stress. Two forms of ECA (phospholipid-linked ECA
(ECAPG), and cyclic ECA (ECACYC)) have different roles related to OM permeability; however, their precise
functions remain unknown, in part, because many steps in their biogenesis are poorly understood.
Our long-term goal is to understand the biogenesis of ECA to facilitate functional studies and identify potential
antimicrobial targets. Specifically, this project aims to elucidate, in Escherichia coli K12, the regulation of and
unknown steps in biogenesis of the forms of ECA contributing to antibiotic resistance. Biochemical reactions are
required for these forms of ECA to be produced and yet the genes responsible for these steps and the regulation
of these steps are largely unknown. The central hypothesis is that ECAPG and ECACYC can be differentiate
through their unique biosynthetic genes and regulatory roles. This hypothesis will be addressed with the following
aims: identify the genes and substrate necessary for ECA to become a phospholipid head group forming ECAPG
using genetic interactions with other biosynthesis pathways (Aim 1); elucidate factors and mechanisms involved
in ECACYC biogenesis using an antibiotic sensitivity suppression phenotype we discovered (Aim 2); and uncover
the mechanisms of the two novel pathways of ECA regulation we discovered (Aim 3). These conceptually
innovative aims will be approached through a blend of high-throughput genomics, genetic screens and
selections, and biochemical techniques. Completion of this project will identify genes and residues important for
biogenesis of ECAPG and ECACYC, which represent targets for development of small molecules weakening the
OM. In addition, this will allow genetic analyses of ECA function, providing insights into Enterobacterales biology.

## Key facts

- **NIH application ID:** 11116447
- **Project number:** 3R01AI155915-04S1
- **Recipient organization:** TEXAS A&M UNIVERSITY
- **Principal Investigator:** Angela Marie Mitchell
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $72,022
- **Award type:** 3
- **Project period:** 2021-06-09 → 2025-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11116447

## Citation

> US National Institutes of Health, RePORTER application 11116447, Biogenesis of cyclic and phospholipid-linked enterobacterial common antigen (3R01AI155915-04S1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/11116447. Licensed CC0.

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